We request funds toward the purchase of cryo-EM sample preparation equipment, including a high pressure freezer (HPF), an automatic freeze substitution system, a cryo-ultramicrotome, a plunge freezing device and the cryo upgrade of an existing transmission electron microscope (TEM) Tecnai T12 (FEI). These instruments will be integrated into a newly re-organized electron microscopy (EM) core facility, the only core facility for researchers at the University of Maryland Baltimore (UMB), University of Maryland Biotechnology Institute (UMBI), University of Maryland Baltimore County (UMBC) and other neighboring research institutes. It is well established that cryo sample preparation techniques offers better antigen preservation and enhanced spatial resolution of ultrastructures. The cryo TEM upgrade, incorporating a cryo sample holder and low dose imaging technologies will allow researchers to observe frozen hydrated biological samples at high resolution, an option that has not been available to researchers at UMB and other surrounding institutes. Many of the major user research projects (Drs. Bavoil, Nataro, Donnenberg, Russell and Bloch) described in this application will utilize immunogold techniques following HPF, freeze substitution and resin embedding at low temperature to enhance antigen detection and ultrastructural preservation. Major users Drs. Donnenberg and Bavoil will use the plunge freeze device to prepare purified protein complexes or microorganisms in vitreous ice and subsequently perform high resolution ultrastructure analysis in the cryo TEM without the use of heavy metal staining. Furthermore, the rapid transfer system used in combination with the HPF allows correlative studies of the same specimen by both light and electron microscopy. This feature complements the capability of live cell imaging of the newly acquired Zeiss Duo laser scanning confocal microscope (purchased in 2008 via SIG 1S10RR02454801, Thomas Blanpied, investigator, major user on this application) and the growing number of research projects studying live cells on the UMB/UMBI campus. Major users Drs. Blanpied and Martin plan to use this feature to enhance their live cell-imaging research. This grant is based upon funded projects for which the cryo sample preparation and cryo EM techniques offer substantial improvements over conventional EM methods for animal cells, whole tissues and microorganisms. Other ongoing projects in this research community will also immediately benefit from the expanded capability of the EM core facility.